Forazolines, compositions thereof and uses thereof

ABSTRACT

An isolated compound of Formula I and salts thereof are provided:wherein X is Cl or Br. A compound isolated from Actinomadura and having a chemical formula of C43H69ClN4O10S2 or C43H69BrN4O10S2 is also provided. Compositions including the compounds and methods of using the compounds to treat fungal infections including those such as Candida are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/146,672, filed on May 4, 2016, which is a continuation of U.S.application Ser. No. 14/299,953, filed on Jun. 9, 2014, now U.S. Pat.No. 9,346,828, issued on May 24, 2016, which claims priority to U.S.Provisional Application No. 61/832,597, filed on Jun. 7, 2013, thecontents of which are incorporated by reference in their entirety intothe present disclosure.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under GM092009 awardedby the National Institutes of Health. The government has certain rightsin the invention.

FIELD OF THE TECHNOLOGY

The present technology relates to a new class of compounds calledForazolines, compositions and methods of use thereof. Specifically, newisolated compounds useful as antifungals are disclosed herein.

SUMMARY

A new compound, called Forazoline A, as well as other Forazolines hasbeen discovered and isolated from Actinomadura, a bacterium collectedfrom a species of sea squirt. Forazoline A has the chemical formulaC₄₃H₆₉ClN₄O₁₀S₂ and is water soluble (e.g., about 5 mg/mL). The isolatedcompound exhibits ¹⁵N NMR peaks at about 140.2 ppm and about 302.6 ppmand an ¹H NMR peak at about 14.58 ppm. The isolated compound furtherexhibits ¹³C NMR peaks at about 76.2 ppm, about 78.2 ppm, about 166.5ppm, about 170.3 ppm, and about 212.0 ppm. The isolated compoundexhibits an IR band at about 1060 cm⁻¹. Pharmaceutical compositionsincluding forazaoline (or pharmaceutically acceptable salts thereof) anda pharmaceutically acceptable carrier are provided. Methods of treatingfungal infections (such as Candida) by administering Forazoline to amammal in need thereof are disclosed.

The foregoing summary is illustrative only and is not intended to be inany way limiting. In addition to the illustrative aspects, embodiments,and features described above, further aspects, embodiments, and featureswill become apparent by reference to the drawings and the followingdetailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an ¹H NMR (600 MHz, CDCl₃) spectrum of Forazoline A.

FIG. 2 shows a ¹³C NMR (125 MHz, CDCl₃) spectrum of Forazoline A.

FIG. 3 shows a gCOSY (600 MHz, CDCl₃) spectrum of Forazoline A.

FIG. 4 shows a gHSQC (600 MHz, CDCl₃) spectrum of Forazoline A.

FIG. 5 shows a gHMBC (600 MHz, CDCl₃) spectrum of Forazoline A.

FIG. 6 shows a ¹H-¹⁵N HMBC (500 MHz, CDCl₃) spectrum of (¹⁵N-labeled)Forazoline A.

FIG. 7 shows a ¹³C NMR (125 MHz, CDCl₃) spectrum of (¹³C-labeled)Forazoline A.

FIG. 8 shows a ¹³C-¹³C COSY (125 MHz, CDCl₃) spectrum of (¹³C-labeled)Forazoline A.

FIG. 9 shows a ¹³C-¹⁵N NMR spectrum of (¹³C-, ¹⁵N-labeled) Forazoline A.

FIG. 10 shows a HRMS spectrum of Forazoline A.

FIG. 11 shows an IR spectrum of Forazoline A.

FIG. 12 shows the fungal membrane permeability of Forazoline A and showsa comparison of the potency of Forazoline A to amphotericin B (AMB).

DETAILED DESCRIPTION

The present technology provides an isolated compound useful for thetreatment of fungal infections such as those caused by the yeastCandida. Thus, in accordance with one aspect, the technology includes acompound isolated from Actinomadura sp. (strain WMMB-499) and having achemical formula of C₄₃H₆₉ClN₄O₁₀S₂ or C₄₃H₆₉BrN₄O₁₀S₂ and saltsthereof, including but not limited to, pharmaceutical salts thereof.

The present technology further provides an isolated compound having achemical formula of C₄₃H₆₉ClN₄O₁₀S₂ (and salts thereof) may exhibit ¹⁵NNMR peaks at about 140.2 ppm and about 302.6 ppm and an ¹H NMR peak atabout 14.58 ppm. In some embodiments, the isolated compound may exhibit¹³C NMR peaks at about 76.2, about 78.2, about 166.5, about 170.3 ppm,and about 212.0 ppm. In other embodiments, the isolated compound havinga chemical formula of C₄₃H₆₉ClN₄O₁₀S₂ (and salts thereof) may exhibit anIR band at about 1060 cm⁻¹. The term “about” will be understood by thoseof skill in the art to include values within ±2% of the stated value.

In another embodiment, the present technology provides an isolatedcompound of Formula I:

and salts thereof, wherein X is Cl or Br.

In some embodiments, the isolated compound (and salts thereof) hasFormula IA, 1B or 1C:

wherein X is Cl or Br.

The compounds described herein may be isolated at various purities,e.g., a purity of at least 60 wt %, at least 70 wt %, at least 80 wt %,at least 90 wt %, at least 95 wt %, at least 96, at least 97 wt %, atleast 98 wt %, at least 99 wt % or at least 99.5 wt %.

Those of skill in the art will appreciate that compounds of the presenttechnology may exhibit the phenomena of tautomerism, conformationalisomerism, geometric isomerism and/or stereoisomerism. As the formuladrawings within the specification and claims can represent only one ofthe possible tautomeric, conformational isomeric, stereoisomeric orgeometric isomeric forms, it should be understood that the technologyencompasses any tautomeric, conformational isomeric, stereoisomericand/or geometric isomeric forms of the compounds having one or more ofthe utilities described herein, as well as mixtures of these variousdifferent forms.

Stereoisomers of compounds (also known as optical isomers) include allchiral, diastereomeric, and racemic forms of a structure, unless thespecific stereochemistry is expressly indicated. Thus, compoundsdisclosed herein include enriched or resolved optical isomers at any orall asymmetric atoms as are apparent from the depictions. Both racemicand diastereomeric mixtures, as well as the individual optical isomerscan be isolated or synthesized so as to be substantially free of theirenantiomeric or diastereomeric partners, and these stereoisomers are allwithin the scope of the present technology.

Salts, including pharmaceutically acceptable salts of the disclosedcompounds are within the scope of the present technology. When thecompound has a basic group, such as, for example, an amine group,pharmaceutically acceptable salts can be formed with inorganic acids(such as hydrochloric acid, sulfuric acid, and phosphoric acid), organicacids (e.g. formic acid, acetic acid, fumaric acid, oxalic acid,tartaric acid, lactic acid, maleic acid, citric acid, succinic acid,malic acid, methanesulfonic acid, benzenesulfonic acid, andp-toluenesulfonic acid) or acidic amino acids (such as aspartic acid andglutamic acid).

In another aspect the present technology provides a pharmaceuticalcomposition including any of the isolated compounds described herein ora pharmaceutical salt thereof, and a pharmaceutically acceptablecarrier. Pharmaceutical compositions of the present technology may beformulated for oral, parenteral, nasal, or topical administration.

In some embodiments, there is provided a pharmaceutical compositionincluding a compound of Formula I, or pharmaceutically acceptable saltthereof, and a pharmaceutically acceptable carrier, wherein Formula Ihas the structure:

wherein X is Cl or Br.

In some embodiments, the pharmaceutical composition includes a compoundof Formula IA, IB, IC, or a pharmaceutically acceptable salt thereof,and a pharmaceutically acceptable carrier, wherein Formula IA has thestructure:

In some embodiments, the pharmaceutical compositions described hereinare formulated for oral, parenteral, nasal, or topical administration.

In another aspect, the present technology provides a method of treatinga fungal infection comprising administering an effective amount of acompound, a salt thereof, or a pharmaceutical composition as describedherein to a mammal in need thereof. The mammal may be, e.g., a human,primate (e.g. monkey, chimpanzee, ape), cat, dog, pig, mouse, rat,horse, sheep, among others. In some embodiments, the mammal is human.The infection may occur, e.g., in the skin, mouth, pharynx, esophagus,toenails, fingernails, and genitalia (including vagina and penis), ormay be systemic, in, e.g., immunocompromised patients. In certainembodiments of the present methods, the fungal infection is caused byCandida, e.g., Candida albicans, Candida enolase, Candida tropicalis,Candida glabrata, Candida krusei, Candida parapsilosis, Candidastellatoidea, Candida parakawsei, Candida lusitaniae, Candidapseudotropicalis, and Candida guilliermondi.

In yet another aspect the present technology provides a compound asdescribed herein, for use in therapy, such as for treatment of fungalinfections. In some embodiments, the fungal infection is caused byCandida, e.g., Candida albicans. In still other embodiments, the presenttechnology provides any of the compounds described herein for use in themanufacture of a medicament for treating a fungal infection. The fungalinfection may be caused by Candida, e.g., Candida albicans.

In another aspect, the present technology provides pharmaceuticalcompositions of the herein-described compounds (including but notlimited to compounds of Formula I, IA, IB, IC, and II) with a secondantifungal agent, e.g., amphotericin B, as well as methods of using thesame. Antifungal agents include drugs which demonstrate clinical benefitin treatment of fungal infections in a mammal, including a human. Insome embodiments, an effective amount of a compound as described herein(including but not limited to compounds of Formulae I, IA, IB, IC, andII), a salt thereof, or a pharmaceutical composition comprising thecompound or salt thereof, and a pharmaceutically acceptable carrier isadministered a mammal in need thereof, wherein amphotericin B isadministered to the mammal in need thereof simultaneously, sequentiallyor separately with a compound as described herein, the salt thereof orthe pharmaceutical composition. While not wishing to be bound by theory,it is believed that the compounds described herein produce theirantifungal activity by affecting the membrane permeability of the fungalcells. In some embodiments the combination of a compound describedherein and a second antifungal agent is synergistic and synergisticallyeffective amounts of the compound and/or second agent may be used. Thatis, lower amounts of the compound and/or agent may be used than would bethe case if the therapeutic effects of the compounds and/or agents weremerely additive. In some embodiments the second agent may be selectedfrom amphotericin B, ketoconazole, terbinafine, nystatin, fluconazole,itraconazole, and voriconazole. In other embodiments the second agent isselected from imidazole-type agents, triazoles-type agents, terbinafine,and nystatin. Imidazole-type antifungal agents contain imidazole as partof their chemical structure, while triazoles-type antifungal agentscontain a triazoles as part of their chemical structure.

“Treating” within the context of the instant technology, means analleviation, in whole or in part, of symptoms associated with a disorderor disease, or slowing, inhibition or halting of further progression orworsening of those symptoms, or prevention or prophylaxis of the diseaseor disorder in a subject at risk for developing the disease or disorder.For example, within the context of treating fungal infections such asthose caused by Candida, successful treatment may include clinicalbenefit, an alleviation of symptoms, such as a reduction or eliminationof redness, itching, discomfort and/or thrush.

As used herein, an “effective amount” of a compound of the presenttechnology refers to an amount of the compound that alleviates, in wholeor in part, symptoms associated with a disorder or disease, or slows orhalts of further progression or worsening of those symptoms, or preventsor provides prophylaxis for the disease or disorder in a subject at riskfor developing the disease or disorder.

The instant technology also provides for compositions and medicamentsincluding a compound disclosed herein and a pharmaceutically acceptablecarrier. Such compositions may be prepared by mixing one or morecompounds of the present technology, pharmaceutically acceptable saltsthereof or stereoisomers thereof, with pharmaceutically acceptablecarriers, excipients, binders, diluents or the like to treat fungalinfections caused by Candida. The compounds and compositions of thepresent technology may be used to prepare formulations and medicamentsthat treat a variety of Candida infections, e.g., Candida albicans. Suchcompositions can be in the form of, for example, granules, powders,tablets, capsules, creams, ointments, syrup, suppositories, injections,emulsions, elixirs, suspensions or solutions. The instant compositionscan be formulated for various routes of administration, for example, byoral, parenteral, topical, injection, rectal, nasal, vaginal, or viaimplanted reservoir. Parenteral or systemic administration includes, butis not limited to, subcutaneous, intravenous, intraperitoneally,intramuscular, intrathecal, intracranial, and intracerebroventricularinjections. The following dosage forms are given by way of example andshould not be construed as limiting the instant technology.

For oral, buccal, and sublingual administration, powders, suspensions,granules, tablets, pills, capsules, gelcaps, and caplets are acceptableas solid dosage forms. These can be prepared, for example, by mixing oneor more compounds disclosed herein, or pharmaceutically acceptable saltsor stereoisomers thereof, with at least one additive such as a starch orother additive. Suitable additives are sucrose, lactose, cellulosesugar, mannitol, maltitol, dextran, starch, agar, alginates, chitins,chitosans, pectins, tragacanth gum, gum arabic, gelatins, collagens,casein, albumin, synthetic or semi-synthetic polymers or glycerides.Optionally, oral dosage forms can contain other ingredients to aid inadministration, such as an inactive diluent, or lubricants such asmagnesium stearate, or preservatives such as paraben or sorbic acid, oranti-oxidants such as ascorbic acid, tocopherol or cysteine, adisintegrating agent, binders, thickeners, buffers, sweeteners,flavoring agents or perfuming agents. Tablets and pills may be furthertreated with suitable coating materials known in the art.

Liquid dosage forms for oral administration may be in the form ofpharmaceutically acceptable emulsions, syrups, elixirs, suspensions, andsolutions, which may contain an inactive diluent, such as water.Pharmaceutical formulations and medicaments may be prepared as liquidsuspensions or solutions using a sterile liquid, such as, but notlimited to, an oil, water, an alcohol, and combinations of these.Pharmaceutically suitable surfactants, suspending agents, emulsifyingagents, may be added for oral or parenteral administration.

As noted above, suspensions may include oils. Such oils include, but arenot limited to, peanut oil, sesame oil, cottonseed oil, corn oil andolive oil. Suspension preparation may also contain esters of fatty acidssuch as ethyl oleate, isopropyl myristate, fatty acid glycerides andacetylated fatty acid glycerides. Suspension formulations may includealcohols, such as, but not limited to, ethanol, isopropyl alcohol,hexadecyl alcohol, glycerol and propylene glycol. Ethers, such as butnot limited to, poly(ethyleneglycol), petroleum hydrocarbons such asmineral oil and petrolatum; and water may also be used in suspensionformulations.

Injectable dosage forms generally include aqueous suspensions or oilsuspensions, which may be prepared using a suitable dispersant orwetting agent and a suspending agent. Injectable forms may be insolution phase or in the form of a suspension, which is prepared with asolvent or diluent. Acceptable solvents or vehicles include sterilizedwater, Ringer's solution, or an isotonic aqueous saline solution.Alternatively, sterile oils may be employed as solvents or suspendingagents. Typically, the oil or fatty acid is non-volatile, includingnatural or synthetic oils, fatty acids, mono-, di- or tri-glycerides.

For injection, the pharmaceutical formulation and/or medicament may be apowder suitable for reconstitution with an appropriate solution asdescribed above. Examples of these include, but are not limited to,freeze dried, rotary dried or spray dried powders, amorphous powders,granules, precipitates, or particulates. For injection, the formulationsmay optionally contain stabilizers, pH modifiers, surfactants,bioavailability modifiers and combinations of these.

Compounds of the present technology also may be formulated as acomposition for topical administration (e.g., vaginal cream). Theseformulations may contain various excipients known to those skilled inthe art. Suitable excipients may include, but are not limited to, cetylesters wax, cetyl alcohol, white wax, glyceryl monostearate, propyleneglycol monostearate, methyl stearate, benzyl alcohol, sodium laurylsulfate, glycerin, mineral oil, water, carbomer, ethyl alcohol, acrylateadhesives, polyisobutylene adhesives, and silicone adhesives.

In some embodiments, the composition is in the form of a vaginal creamcontaining the composition of matter as set forth herein present in anonliquefying base. The nonliquefying base may contain various inactiveingredients such as, for example, cetyl esters wax, cetyl alcohol, whitewax, glyceryl monostearate, propylene glycol monostearate, methylstearate, benzyl alcohol, sodium lauryl sulfate, glycerin, and mineraloil. Such composition may be formulated similar to PREMARIN® VaginalCream made commercially available by Wyeth-Ayerst Laboratories.

Dosage units for rectal administration may be prepared in the form ofsuppositories which may contain the composition of matter in a mixturewith a neutral fat base, or they may be prepared in the form ofgelatin-rectal capsules which contain the active substance in a mixturewith a vegetable oil or paraffin oil.

Compounds of the present technology may be administered to the lungs byinhalation through the nose or mouth. Suitable pharmaceuticalformulations for inhalation include solutions, sprays, dry powders, oraerosols containing any appropriate solvents and optionally othercompounds such as, but not limited to, stabilizers, antimicrobialagents, antioxidants, pH modifiers, surfactants, bioavailabilitymodifiers and combinations of these. Formulations for inhalationadministration contain as excipients, for example, lactose,polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate. Aqueousand nonaqueous aerosols are typically used for delivery of inventivecompounds by inhalation.

Ordinarily, an aqueous aerosol is made by formulating an aqueoussolution or suspension of the compound together with conventionalpharmaceutically acceptable carriers and stabilizers. The carriers andstabilizers vary with the requirements of the particular compound, buttypically include nonionic surfactants (Tweens, Pluronics, orpolyethylene glycol), innocuous proteins like serum albumin, sorbitanesters, oleic acid, lecithin, amino acids such as glycine, buffers,salts, sugars or sugar alcohols. Aerosols generally are prepared fromisotonic solutions. A nonaqueous suspension (e.g., in a fluorocarbonpropellant) can also be used to deliver compounds of the presenttechnology.

Aerosols containing compounds for use according to the presenttechnology are conveniently delivered using an inhaler, atomizer,pressurized pack or a nebulizer and a suitable propellant, e.g., withoutlimitation, pressurized dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, nitrogen, air, or carbon dioxide. In the caseof a pressurized aerosol, the dosage unit may be controlled by providinga valve to deliver a metered amount. Capsules and cartridges of, forexample, gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch. Delivery of aerosols of the present technologyusing sonic nebulizers is advantageous because nebulizers minimizeexposure of the agent to shear, which can result in degradation of thecompound.

For nasal administration, the pharmaceutical formulations andmedicaments may be a spray, nasal drops or aerosol containing anappropriate solvent(s) and optionally other compounds such as, but notlimited to, stabilizers, antimicrobial agents, antioxidants, pHmodifiers, surfactants, bioavailability modifiers and combinations ofthese. For administration in the form of nasal drops, the compounds maybe formulated in oily solutions or as a gel. For administration of nasalaerosol, any suitable propellant may be used including compressed air,nitrogen, carbon dioxide, or a hydrocarbon based low boiling solvent.

Besides those representative dosage forms described above,pharmaceutically acceptable excipients and carriers are generally knownto those skilled in the art and are thus included in the instant presenttechnology. Such excipients and carriers are described, for example, in“Remingtons Pharmaceutical Sciences” Mack Pub. Co., New Jersey (1991),which is incorporated herein by reference.

The formulations of the present technology may be designed to beshort-acting, fast-releasing, long-acting, and sustained-releasing asdescribed below. Thus, the pharmaceutical formulations may also beformulated for controlled release or for slow release.

The instant compositions may also comprise, for example, micelles orliposomes, or some other encapsulated form, or may be administered in anextended release form to provide a prolonged storage and/or deliveryeffect. Therefore, the pharmaceutical formulations and medicaments maybe compressed into pellets or cylinders and implanted intramuscularly orsubcutaneously as depot injections or as implants such as stents. Suchimplants may employ known inert materials such as silicones andbiodegradable polymers.

Specific dosages may be adjusted depending on conditions of disease, theage, body weight, general health conditions, sex, and diet of thesubject, dose intervals, administration routes, excretion rate, andcombinations of drugs. Any of the above dosage forms containingeffective amounts are well within the bounds of routine experimentationand therefore, well within the scope of the instant technology.

A therapeutically effective amount of a compound of the presenttechnology may vary depending upon the route of administration anddosage form. Effective amounts of such compounds typically fall in therange of about 0.01 up to about 100 mg/kg/day, or about 0.05 to about 50mg/kg/day, and more typically in the range of about 0.1 up to 5mg/kg/day. Typically, the compound or compounds of the instanttechnology are selected to provide a formulation that exhibits a hightherapeutic index. The therapeutic index is the dose ratio between toxicand therapeutic effects and can be expressed as the ratio between LD₅₀and ED₅₀. The LD₅₀ is the dose lethal to 50% of the population and theED₅₀ is the dose therapeutically effective in 50% of the population. TheLD₅₀ and ED₅₀ are determined by standard pharmaceutical procedures inanimal cell cultures or experimental animals.

All publications, patent applications, issued patents, and otherdocuments referred to in this specification are herein incorporated byreference as if each individual publication, patent application, issuedpatent, or other document was specifically and individually indicated tobe incorporated by reference in its entirety. Definitions that arecontained in text incorporated by reference are excluded to the extentthat they contradict definitions in this disclosure.

The present technology is further illustrated by the following examples,which should not be construed as limiting in any way.

EXAMPLES

General Experimental Procedures

Optical rotations were measured on a Perkin-Elmer 241 Polarimeter.Ultraviolet (UV) spectra were recorded on an Aminco/OLIS UV-Visspectrophotometer. Infrared (IR) spectra were measured with a BrukerEquinox 55/S FT-IR spectrophotometer. Nuclear magnetic resonance (NMR)spectra were obtained in CDCl₃ with a Bruker Avance 600 MHz spectrometerequipped with a 1.7 mm ¹H{₁₃C/¹⁵N} cryoprobe and a Bruker Avance 500 MHzspectrometer equipped with a ¹³C/¹⁵N{¹H} cryoprobe. High resolution massspectrometry (HRMS) data were acquired with a Bruker MaXis 4G QTOF massspectrometer. Reverse phase high pressure liquid chromatography (RPHPLC) was performed using a Shimadzu Prominence HPLC system with aPhenomenex Luna C₁₈ column (250×10 mm, 5 μm) and a Gilson HPLC systemwith a Phenomenex Gemini C₁₈ column (100×30 mm, 5 μm).

Example 1 Isolation of Forazoline a from Bacteria Associated withAscidiacea

Biological Material. Ascidian specimens were collected in the FloridaKeys (24° 39.591′, 81° 25.217′). A voucher specimen (FLK10-5-6) forEcteinascidia turbinata (Herdman, 1880) is housed at the University ofWisconsin-Madison. For cultivation, a sample of ascidian (1 cm³) wasrinsed with sterile seawater, macerated using a sterile pestle in amicro-centrifuge tube, and dilutions were made in sterile seawater, withvortexing between steps to separate bacteria from heavier tissues.Dilutions were separately plated on three media: ISP2, R2A, and M4. Eachmedium was supplemented with 50 μg/mL cycloheximide and 25 μg/mLnalidixic acid. Plates were incubated at 28° C. for at least 28 days.Actinomadura sp. strain WMMB-499 was isolated from ISP2 medium.

Sequencing. 16S rDNA sequencing was conducted as previously described(Wyche, T. P.; Hou, Y.; Braun, D.; Cohen, H. C.; Xiong, M. P.; Bugni, T.S. J. Org. Chem. 2011, 76, 6542-6547). WMMB499 was identified as anActinomadura sp. and demonstrated 99% sequence similarity toActinomadura sp. 13679C (accession number EU741239). The 16S sequencefor WMMB499 was deposited in GenBank (accession number JX101467).

Fermentation, Extraction, and Isolation. Two 10 mL seed cultures (25×150mm tubes) in medium ASW-A (20 g soluble starch, 10 g glucose, 5 gpeptone, 5 g yeast extract, 5 g CaCO₃ per liter of artificial seawater)were inoculated with strain WMMB499 and shaken (200 RPM, 28° C.) forseven days. Two hundred fifty mL baffled flasks (3×50 mL) containingASW-A were inoculated with 1 mL seed culture and were shaken (200 RPM,28° C.) for seven days. Two-liter flasks (6×500 mL) containing mediumASW-A with Diaion HP20 (4% by weight) were inoculated with 25 mL fromthe 50 mL culture and shaken (200 RPM, 28° C.) for seven days. FilteredHP20 and cells were washed with H₂O and extracted with acetone. Theacetone extract (3.2 g) was subjected to liquid-liquid partitioningusing 30% aqueous methanol (MeOH) and CHCl₃ (1:1). The CHCl₃-solublepartition (2.2 g) was fractionated by Sephadex LH20 columnchromatography (CHCl₃:MeOH, 1:1). Fractions containing forazoline A(compound of Formula I, X=Cl) were subjected to RP HPLC (10/90% to100/0% MeOH—H₂O containing 0.1% acetic acid, 22 min) using a PhenomenexGemini C18 column (100×30 mm, 5 μm), yielding forazoline A (20.1 mg,t_(R) 12.5 min). For ¹³C and ¹⁵N incorporation, the same procedure wasused (1×250 mL) with labeled medium ASW-A (20 g soluble starch, 10 gU¹³C-glucose, 2.5 g peptone, 2.5 g yeast extract, 5 g ¹⁵NH₄Cl, 5 g CaCO₃per liter of artificial seawater). For the incorporation of bromine andthe production of forazoline B (compound of Formula I, X=Br), the sameprocedure was used with two-liter flasks (2×500 mL) containing mediumASW-A (with an increase of KBr from 0.1 g/L to 10 g/L and elimination of20 g/L NaCl).

Acetylation. Forazoline A (1.0 mg) was dissolved in 150 μL pyridine, and50 μL acetic anhydride was added to the solution. The solution wasstirred at room temperature for 24 hours. The product was dried underargon.

Example 2 Biological Activity of Forazoline A

Animals. Six-week-old ICR Swiss specific-pathogen-free female miceweighing 23 to 27 g were used for all studies. Animals were maintainedin accordance with the American Association for Accreditation ofLaboratory Care criteria. Animal studies were approved by the Universityof Wisconsin Animal Care Committee.

Infection model. A neutropenic, murine, disseminated candidiasis modelwas used for the treatment studies. Mice were rendered neutropenic(polymorphonuclear cell counts of <100 mm³) by injectingcyclophosphamide subcutaneously 4 days before infection (150 mg/kg ofbody weight) and 1 day before infection (100 mg/kg). Candida albicans K1was subcultured on Sabouraud dextrose agar (SDA) 24 h prior toinfection. The inocula were prepared by placing three to five coloniesinto 5 ml of sterile 0.15 M NaCl warmed to 35° C. The final inoculum wasadjusted to 0.6 transmittance at 530 nm. Fungal counts of the inoculadetermined by viable counts on SDA were 5.63±0.38 log₁₀ CFU/mL(mean±standard deviation).

Disseminated infection with C. albicans organisms was produced byinjection of 0.1 ml of the inoculum via the lateral tail vein 2 h priorto the start of antifungal therapy. At the end of the study period (8h), animals were sacrificed by CO₂ asphyxiation. After sacrifice thekidneys of each mouse were immediately removed and placed in sterile0.15 M NaCl at 4° C. The organs were homogenized and then seriallydiluted 1:10. Aliquots were plated onto SDA for viable fungal colonycounts after incubation for 24 h at 35° C. The lower limit of detectionwas 100 CFU/kidney. The results are expressed as the mean and standarddeviation of the log₁₀ CFU/kidney from three mice.

Drug Treatment. Groups of three mice were treated with either anintravenous or intraperitoneal single of forazoline A at 0.312, 1.25,and 5 mg/kg. The intravenous dose was given by the lateral tail vein viaa 200 μL infusion. The intraperitoneal dose was administered in 500 μLvolume. Control mice were treated with saline. Groups of mice weresacrificed at the start of therapy and 8 hours after therapy fordetermination of organism burden in the kidney as described above.

Results. Forazoline demonstrated in vivo efficacy in neutropenic(immunocompromised) mice in a disseminated candidiasis model againstCandida albicans K1. Mice were treated with the compound atconcentrations 2.5, 0.78, and 0.125 mg/kg. After 8 hours, the micetreated with the compound showed a decrease in greater than 1 log₁₀cfu/kidney (1.5+/−0.12) reduction in organism burden compared to controlmice. No toxic effects from the compound were apparent.

Example 3 Yeast Membrane Permeability

Forazoline A demonstrated a novel mechanism of action, as determined bychemical genomic profiling with the yeast Saccharomyces cerevisiae. Thismethod has been used to explore the mechanism of action for bioactivecompounds, including natural products. Forazoline A was screened againstover four thousand deletion mutant yeast strains, genomic DNA wasextracted, and mutant-specific DNA barcodes were amplified and sequencedby Illumina sequencing. Forazoline A sensitive and resistant mutantswere determined by quantification of DNA-barcodes, which provided achemical genomic profile used to evaluate the mechanism of action.

The top sensitive mutant strains (P<0.0001) were significantly enrichedfor genes involved in phospholipid translocation (GO: 0045332,P=0.0009). This enrichment was driven by sensitive mutants withdeletions of the genes LEM3 and FPK1. Enzymes Lem3p formed complexeswith Dnf1p/Dnf2p, which is responsible for maintaining phospholipidasymmetry in membranes while Fpk1p is a Ser/Thr protein kinase thatregulates Lem3p-Dnf1p/Dnf2p (Dnf1p is a phospholipid translocase). Thedata suggests that forazoline A either directly affects phospholipids orinteracts with a protein target that complements the activity of theLem3p complexes. An important aspect of the data was that the mutantLEM3-Δ was not among the most sensitive strains for caspofungin,fluconazole, or amphotericin, which suggests that forazoline A has aunique mechanism of action from known antifungal agents.

The membrane integrity of yeast cells treated with forazoline A wasinvestigated through the evaluation of membrane permeability. As shownin FIG. 12, the results demonstrated that forazoline A caused a dosedependent permeabilization of fungal membranes after 4 h of treatment,but at the same concentration as amphotericin B (AMB) (125 μg/mL) wasless potent.

Since chemical genomics suggested that forazoline A had a differentmechanism compared to AMB, synergy studies were conducted. Forazoline Ashowed synergy when tested with AMB indicating a parallel and/orcomplementary mechanism of action. The data indicated that membraneintegrity was affected by forazoline A.

Example 4 Structure Elucidation

Analytical data were gathered for forazoline A, including opticalrotation, IR, HRMS and NMR spectra.

Forazoline A (compound of Formula I, X=Cl): yellow solid; [α]²⁵ _(D)+300(c 0.6, MeOH); UV (MeOH) λ_(max) (log ε) 205 (4.11), 222 (3.91), 254(3.74), 274 (3.54), 406 (4.05) nm; IR (ATR) (see FIG. 11) U_(max)2940,2359, 1733, 1533, 1472, 1060 cm⁻¹; ¹H and ¹³C NMR (See Table 1 and FIGS.1, 2 and 7); HRMS [M+H]⁺ m/z 901.4093 (calc'd for C₄₃H₇₀ClN₄O₁₀S₂,901.4216) (see FIG. 10).

TABLE 1 ¹H and ¹³C NMR data (600 MHz, CDCl₃) Forazoline A δ_(c), δ_(H)Position mult. (J in Hz) COSY* HMBC** 1 40.9, CH₃ 2.22, s 2, 3 2 40.9,CH₃ 2.22, s 1, 3 3 65.0, CH 2.26, m 4, 6 5 4 74.2, CH 3.47, m 3, 5 5, 85 19.4, CH₃ 1.28, d (6.3) 4 3, 4 6 18.7, CH₂ 1.48, m 3, 7 3, 7, 8 1.85,m 7 32.6, CH₂ 1.65, m 6, 8 1.95, m 8 97.9, CH 4.76, dd (1.9, 7 7, 9 9.4)9 92.6, C 10 22.8 CH₃ 1.75. s 9, 10, 44 11 78.8, CH 4.19, d (10.1) 11 1212 27.3, CH₂ 1.30, m 10, 12 10, 14 1.69, m 13 32.1, CH₂ 2.05, m 11, 1611, 14, 16 2.16, m 14 136.0, C 15 19.9, CH₃ 1.63, s 16 12, 14, 16 16122.4, CH 5.08, d (9.4) 12, 15, 17 13, 15 17 47.8, CH 2.33, q (9.8) 16,18 14, 16, 18, 31, 32 18 73.4, CH 4.04, m 17, 27 16, 19, 27 19 101.8, CH4.91, dd (1.3, 20 18, 20 9.1) 20 31.7, CH₂ 1.30, m 19, 21 19, 21 2118.7, CH₂ 1.58, m 20, 22 1.81, m 22 65.4, CH 2.15, m 21, 25 23 40.9, CH₃2.22, s 22, 24 24 40.9, CH₃ 2.22, s 22, 23 25 74.3, CH 3.48, m 22, 2619, 26 26 19.3, CH₃ 1.26, d (8.8) 25 22, 25 27 34.7. CH₂ 1.90, m 18, 2817, 29 2.08, m 28 78.2, CH 5.33, t (2.7) 27 18, 29, 30, 31, 33 29 75.9,C 30 22.8, CH₃ 1.53, s 29, 31 31 41.4, CH 1.90, m 32 16, 17, 29, 30, 3232 13.4, CH₃ 0.76, d (6.9) 31 17, 29, 31 33 171.6, C 34 76.3, CH 5.50,dd (4.1, 35 33, 35, 36, 10.4) 38, 40 35 38.8, CH₂ 3.80, t (11.2) 34 33,34 3.94, dd (4.1, 11.3) 36 170.3, C 38 76.2, C 39 24.8, CH₃ 1.59, s 36,37, 39 40 94.4, CH 5.04, s NH 37, 38, 41, 43 41 57.2, CH₃ 3.40, s 39 42166.5, C 43-NH 14.58, s 39 37, 39, 41, 43 44 106.4, CH 7.30, s 39, 41 45212.0, C *See FIG. 3. **See also FIGS. 5 and 6.

Discussion: HRMS supported the molecular formula of C₄₃H₆₉ClN₄O₁₀S₂ forforazoline A (compound of Formula I). Extensive 1D and 2D NMR data(Table 1) were analyzed to establish the majority of the planarstructure, but the presence of several quaternary centers preventedcomplete elucidation of the structure. The carbon backbone of thestructure was determined by increasing the ¹³C abundance withuniformly-labeled ¹³C glucose and acquiring a ¹³C-¹³C gCOSY.Fermentation of WMMB499 in ASW-A with ¹³C-labeled glucose and subsequentpurification (see above), yielded forazoline A with nearly 75% ¹³Cincorporation. The ¹³C-¹³C gCOSY was acquired in 30 minutes on 7.0 mgforazoline A and allowed for complete assignment of the carbon backbone(see FIG. 8).

Despite knowing the carbon backbone, the entire structure could not beelucidated due to a portion of the structure composed of several ringsthat contained multiple heteroatoms—oxygen, nitrogen, and sulfur. The¹³C chemical shifts of several carbon atoms attached to heteroatoms didnot conclusively indicate which heteroatoms were attached to eachcarbon. Therefore, a method was pursued to determine the ¹³C-¹⁵Nconnectivity. Fermentation of 250 mL of WMMB499 in a variation of ASW-A,containing ¹⁵NH₄Cl and uniformly-labeled ¹³C-glucose (see above),allowed for the production of ¹³C- and ¹⁵N-labeled forazoline A. A 2DNMR experiment was then developed to determine the ¹³C-¹⁵N connectivity(see FIG. 9). The spectrum revealed that N-43 (δ_(N) 140.2 ppm) wasattached to C-42 (δ_(C) 166.5) and C-38 (δ_(C) 76.2 ppm), and N-37(δ_(N) 302.6 ppm) was attached to C-36 (δ_(C) 170.3 ppm) and C-34 (δ_(C)78.2 ppm); the spectrum also confirmed the presence of two N-dimethylgroups. The downfield shift of N-37, combined with the knowledge that itwas connected to only two carbons, suggested the presence of an imine.

HRMS of forazoline A in CD₃OD and D₂O revealed the presence of twoexchangeable protons. Acquisition of a ¹H-¹⁵N HSQC (FIG. 4) allowed usto conclude that one of the exchangeable protons was an amine (δ_(H)14.58, δ_(N) 170.3). To determine the location of the other exchangeableproton, forazoline A was acetylated. Two major products, with one andtwo acetyl units, were formed, and acquisition of 1D and 2D NMR,determined that the other exchangeable proton was attached to aheteroatom at C-11. The chemical shift (δ_(C) 78.8) at C-11 determinedthat exchangeable was a hydroxyl.

The HRMS isotopic distribution and molecular formula suggested thepresence of one chlorine atom. To determine the location of the chlorineatom, the amount of KBr in fermentation medium ASW-A was increased from0.1 g/L to 10 g/L to produce a brominated analog, forazoline B (compoundof Formula I, X=Br). HRMS of forazoline B supported the molecularformula of C₄₃H₆₉BrO₁₀N₄S₂. A comparison of the ¹H and ¹³C NMR shifts offorazolines A and B showed that the chemical shifts of H-28, H-31, C-28,and C-31 shifted downfield, and C-29 shifted upfield in forazoline B. Noother significant changes in chemical shifts between forazoline A and Bexisted. Combining this knowledge with the fact that C-29 was a methineattached to two carbons, allowed us to conclude that the halogen atomwas located at C-29.

The presence of the sulfoxide was determined by a combination of IR andDFT calculations. Sulfoxides typically demonstrate an IR band between1015-1060 cm⁻¹. A band at 1060 cm⁻¹ in the IR spectrum of forazoline Asuggested the presence of a sulfoxide. Additionally, the aforementionedNMR data, which provided ¹³C-¹³C and ¹³C-¹⁵N connectivity, limited thenumber of locations for this oxygen. In order to determine which sulfuratom in the molecule contained the sulfoxide, molecular modeling and DFTcalculations were used to calculate theoretical NMR shifts, which couldthen be compared to the experimental data. Molecular modeling and DFTcalculations were performed as previously described (Wyche, T. P.; Hou,Y.; Braun, D.; Cohen, H. C.; Xiong, M. P.; Bugni, T. S. J. Org. Chem.2011, 76, 6542-6547 (incorporated by reference herein)). The DP4probability method allowed for comparison of the theoretical NMR shiftsof the two potential structures with the experimental data. Using all¹³C NMR shifts, the DP4 probability calculated that the theoretical NMRdata for the sulfoxide attached to C-39 and C-41 matched theexperimental NMR data better than the theoretical NMR data for thesulfoxide attached to C-35 and C-36 (See Table 1 and FIGS. 2, and 7-9(¹³C NMR shifts)).

EQUIVALENTS

The present disclosure is not to be limited in terms of the particularembodiments described in this application. Many modifications andvariations can be made without departing from its spirit and scope, aswill be apparent to those skilled in the art. Functionally equivalentmethods and apparatuses within the scope of the disclosure, in additionto those enumerated herein, will be apparent to those skilled in the artfrom the foregoing descriptions. Such modifications and variations areintended to fall within the scope of the appended claims. The presentdisclosure is to be limited only by the terms of the appended claims,along with the full scope of equivalents to which such claims areentitled. It is to be understood that this disclosure is not limited toparticular methods, reagents, compounds compositions or biologicalsystems, which can, of course, vary. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the likeinclude the number recited and refer to ranges which can be subsequentlybroken down into subranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember. Thus, for example, a group having 1-3 cells refers to groupshaving 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers togroups having 1, 2, 3, 4, or 5 cells, and so forth.

While various aspects and embodiments have been disclosed herein, otheraspects and embodiments will be apparent to those skilled in the art.The various aspects and embodiments disclosed herein are for purposes ofillustration and are not intended to be limiting, with the true scopeand spirit being indicated by the following claims.

We claim:
 1. A pharmaceutical composition comprising an effective amountof a compound of Formula I:

or pharmaceutically acceptable salt thereof for treating a fungalinfection in a mammal, wherein X is Cl or Br; and a pharmaceuticallyacceptable carrier.
 2. The pharmaceutical composition of claim 1,formulated for oral, parenteral, nasal, or topical administration. 3.The pharmaceutical composition of claim 1, wherein the mammal is human.4. The pharmaceutical composition of claim 1, further comprisingamphotericin B, ketoconazole, terbinafine, nystatin, fluconazole,itraconazole, or nystatin.
 5. The pharmaceutical composition of claim 1,wherein the compound of Formula I has the Formula IA:

wherein X is Cl or Br.
 6. The pharmaceutical composition of claim 5,wherein the mammal is human.
 7. The pharmaceutical composition of claim1, wherein the compound of Formula I has the Formula IB:


8. The pharmaceutical composition of claim 7, wherein the mammal ishuman.
 9. The pharmaceutical composition of claim 1, wherein thepharmaceutical composition comprises an effective amount of the compoundor pharmaceutically acceptable salt thereof for treating a Candidainfection.
 10. The pharmaceutical composition of claim 9, wherein themammal is human.
 11. The pharmaceutical composition of claim 1, whereinthe pharmaceutical composition comprises an effective amount of thecompound or pharmaceutically acceptable salt thereof for treating aCandida albicans infection in a human.
 12. A pharmaceutical compositioncomprising an effective amount of a compound of Formula I:

or pharmaceutically acceptable salt thereof for treating a fungalinfection in a mammal, wherein X is Cl or Br; and an isotonic aqueoussolution.
 13. The pharmaceutical composition of claim 12, formulated forparenteral administration.
 14. The pharmaceutical composition of claim12, wherein the mammal is human.
 15. The pharmaceutical composition ofclaim 12, wherein the compound of Formula I has the Formula IA:

wherein X is Cl or Br.
 16. The pharmaceutical composition of claim 12,wherein the compound of Formula I has the Formula IB:


17. The pharmaceutical composition of claim 12, wherein thepharmaceutical composition comprises an effective amount of the compoundor pharmaceutically acceptable salt thereof for treating a Candidainfection.
 18. The pharmaceutical composition of claim 12, wherein thepharmaceutical composition comprises an effective amount of the compoundor pharmaceutically acceptable salt thereof for treating a Candidaalbicans infection.
 19. A pharmaceutical composition comprising aneffective amount of a compound of Formula I:

or pharmaceutically acceptable salt thereof for treating a fungalinfection in a mammal, wherein X is Cl or Br; and one or more of cetylesters wax, cetyl alcohol, white wax, glyceryl monostearate, propyleneglycol monostearate, methyl stearate, benzyl alcohol, sodium laurylsulfate, glycerin, mineral oil, carbomer, ethyl alcohol, acrylateadhesives, polyisobutylene adhesives, and silicone adhesives.
 20. Thepharmaceutical composition of claim 19, wherein the mammal is human. 21.The pharmaceutical composition of claim 19, further comprisingamphotericin B, ketoconazole, terbinafine, nystatin, fluconazole,itraconazole, or nystatin.